Sulfate Metabolites Research Articles

Alternative long-term markers for the detection of methyltestosterone misuse

Methyltestosterone (MT) is one of the most frequently detected anabolic androgenic steroids in doping control analysis. MT misuse is commonly detected by the identification of its two main metabolites excreted as glucuronide conjugates, 17α-methyl-5α-androstan-3α,17β-diol and 17α-methyl-5β-androstan-3α,17β-diol. The detection of these metabolites is normally performed by gas chromatography–mass spectrometry, after previous hydrolysis with β-glucuronidase enzymes, extraction and derivatization steps. The aim of the present work was to study the sulphate fraction of MT and to evaluate their potential to improve the detection of the misuse of the drug in sports. MT was administered to healthy volunteers and urine samples were collected up to 30days after administration. After an extraction with ethyl acetate, urine extracts were analysed by liquid chromatography tandem mass spectrometry using electrospray ionisation in negative mode by monitoring the transition m/z 385 to m/z 97. Three diol sulphate metabolites (S1, S2 and S3) were detected. Potential structures for these metabolites were proposed after solvolysis and mass spectrometric experiments: S1, 17α-methyl-5β-androstan-3α,17β-diol 3α-sulphate; S2, 17β-methyl-5α-androstan-3α,17α-diol 3α-sulphate; and S3, 17β-methyl-5β-androstan-3α,17α-diol 3α-sulphate. Synthesis of reference compounds will be required in order to confirm the structures. The retrospectivity of these sulphate metabolites in the detection of MT misuse was compared with the obtained with previously described metabolites. Metabolite S2 was detected up to 21days after MT administration, improving between 2 and 3 times the retrospectivity of the detection compared to the last long-term metabolite of MT previously described, 17α-hydroxy-17β-methylandrostan-4,6-dien-3-one.

Urinary conjugated α-tocopheronolactone—a biomarker of oxidative stress in children with type 1 diabetes

Increased oxidative stress has been implicated in both the onset and the progression of diabetes mellitus and its complications. The development of easy to measure biomarkers of oxidative stress would, therefore, help in determining in a prospective manner the impact of glycemic control on oxidative stress and macrovascular disease in patients with diabetes. We report the development and validation of a novel method to directly measure the urinary concentrations of the conjugated metabolites of vitamin E (α-tocopherol) and investigate whether the oxidized metabolite α-tocopheronolactone (α-TL) could be used as a biomarker of oxidative stress in children with type 1 diabetes. A novel method using liquid chromatography–tandem mass spectrometry was developed and used to measure directly and rapidly the urinary concentrations of the glucuronidated and sulfated metabolites of α-tocopherol in 32 young patients with type 1 diabetes and age-matched controls. The mean concentrations of the glucuronidated and sulfated conjugates of α-TL were all highly significantly increased in the children with type 1 diabetes (p<0.001). The results suggest that the measurement of the urinary concentrations of α-TL conjugates may provide a useful biomarker of oxidative stress in diabetes and possibly in other clinical conditions in which oxidative stress has been implicated.

Mycobacterium Sulfur Metabolism and Implications for Novel Drug Targets

New antibiotic targets are urgently needed to tackle the multidrug resistant and latent Mycobacterium tuberculosis, the causative agent of the most formidable infectious disease tuberculosis. Sulfur metabolism is essential for the survival and virulence of many pathogens including M. tuberculosis. The absence of most genes involved in microbial sulfur metabolism in human beings suggests abundant novel potential antibiotic targets in pathogen sulfur metabolism. In this article, a comparative genomic landscape of Mycobacterium sulfur metabolism, such as the uptake, activation, and reduction of sulfate and allied enzymes, the biosynthesis pathway of some sulfated metabolites, and the enzymes involved in these pathways were presented. Novel clues for antibiotic targets are put forward.

Dietary resveratrol does not delay engraftment, sensitize to vincristine or inhibit growth of high-risk acute lymphoblastic leukemia cells in NOD/SCID mice

Acute lymphoblastic leukemia (ALL) with translocation t(4;11) is a high-risk leukemia found in 60–85% of infants with ALL and is often refractory to conventional chemotherapeutics after relapse. To evaluate the efficacy of dietary resveratrol in vivo, 5-week-old NOD.CB17-Prkdcscid/J mice were fed a control diet or a diet containing 0.2% w/w resveratrol. After 3 weeks of dietary treatment, mice were engrafted with the human t(4;11) ALL line SEM by tail vein injection. Engraftment was monitored by evaluating the presence of human CD19+ cells in peripheral blood using flow cytometry. Relative to control diet, dietary resveratrol did not delay the engraftment of the leukemia cells. To determine if dietary resveratrol could increase efficacy of a chemotherapeutic agent, vincristine was injected intraperitoneally into leukemic mice fed the control or supplemented diet. Survival curves and monitoring the percentage of human leukemia cells in peripheral blood showed that resveratrol did not inhibit leukemia cell growth or influence the activity of vincristine. Mass spectrometric analysis of mouse serum revealed that the majority of resveratrol was present as glucuronidated and sulfated metabolites. These data do not support the concept that dietary resveratrol has potential as a preventative agent against the growth of high-risk t(4;11) ALL.

The Arabidopsis thylakoid ADP/ATP carrier TAAC has an additional role in supplying plastidic phosphoadenosine 5'-phosphosulfate to the cytosol.

3'-Phosphoadenosine 5'-phosphosulfate (PAPS) is the high-energy sulfate donor for sulfation reactions. Plants produce some PAPS in the cytosol, but it is predominantly produced in plastids. Accordingly, PAPS has to be provided by plastids to serve as a substrate for sulfotransferase reactions in the cytosol and the Golgi apparatus. We present several lines of evidence that the recently described Arabidopsis thaliana thylakoid ADP/ATP carrier TAAC transports PAPS across the plastid envelope and thus fulfills an additional function of high physiological relevance. Transport studies using the recombinant protein revealed that it favors PAPS, 3'-phosphoadenosine 5'-phosphate, and ATP as substrates; thus, we named it PAPST1. The protein could be detected both in the plastid envelope membrane and in thylakoids, and it is present in plastids of autotrophic and heterotrophic tissues. TAAC/PAPST1 belongs to the mitochondrial carrier family in contrast with the known animal PAPS transporters, which are members of the nucleotide-sugar transporter family. The expression of the PAPST1 gene is regulated by the same MYB transcription factors also regulating the biosynthesis of sulfated secondary metabolites, glucosinolates. Molecular and physiological analyses of papst1 mutant plants indicate that PAPST1 is involved in several aspects of sulfur metabolism, including the biosynthesis of thiols, glucosinolates, and phytosulfokines.

Biodegradation of endosulfan isomers in broth culture and soil microcosm by Pseudomonas fluorescens isolated from soil

Bacterial isolates from endosulfan-contaminated soil were grown in minimal medium and screened for endosulfan degradation. The isolate which used endosulfan and showed maximum growth was selected for detailed study. Maximum degradation in shake flask culture by Pseudomonas fluorescens was 92.80% of α and 79.35% of β endosulfan isomers in 15 days at 20 mg/L concentration, followed by 50 and 100 mg/L, while the corresponding values in static condition were 69.15 and 51.39%, respectively. Endosulfan concentration degradation declined significantly at 50 and 100 mg/L. Concomitant to degradation, release of chloride ion exhibited positive relation, while pH decreased from 7.0 to 4.53 in agitating and 7.0–5.18 in static condition. The soil microcosm study revealed maximum endosulfan degradation in sterilized soil amended with P. fluorescens. Endosulfan diol and endosulfan ether were among the products of endosulfan metabolism in broth culture, but only endosulfan ether was detected in the soil microcosm. Endosulfan sulphate, a persistent and toxic metabolite of endosulfan, was not detected in either case. The study showed that P. fluorescens could be used effectively for bioremediation of the pesticide contaminated sites.

A novel enzyme‐dependent melatonin metabolite in humans

Exogenous melatonin is widely used in humans for multiple pharmacologic purposes. The metabolic pathways of melatonin reflect the fate and functions of melatonin in vivo. This study was designed to re-profile melatonin metabolism in humans using a metabolomic approach. In the urine of healthy subjects treated with 10 mg melatonin, sulfate- or glucuronide-conjugated metabolites of melatonin were detected, including 6-hydroxymelatonin sulfate, 6-hydroxymelatonin glucuronide, N-acetylserotonin glucuronide, N-acetylserotonin sulfate, and an unknown sulfated metabolite (X). The molecular weight of metabolite X was 14 Da smaller than 6-hydroxymelatonin sulfate, but 16 Da larger than N-acetylserotonin sulfate. Further studies suggest that metabolite X was produced via O-demethylation, 6-hydroxylation, and sulfation. The antioxidant products of melatonin, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine and N(1)-acetyl-5-methoxy-kynuramine, were not detected in human urine. In summary, this study provided a global view of melatonin metabolism in humans and extended our knowledge of enzyme-dependent pathways of melatonin metabolism.

Delipidating effect of resveratrol metabolites in 3T3‐L1 adipocytes

Due to the low bioavailability of resveratrol, determining whether its metabolites exert any beneficial effect is an interesting issue. 3T3-L1 maturing pre-adipocytes were treated from day 0 to day 8 of differentiation and mature adipocytes for 24 h on day 12 with 1, 10, or 25μM of resveratrol or its metabolites. Triacylglycerols were assessed by spectrophotometry and gene expression by real time RT-PCR. Resveratrol, trans-resveratrol-4'-O-glucuronide and trans-resveratrol-3-O-sulfate reduced triacylglycerol content in maturing pre-adipocytes at 25μM. In mature adipocytes, both resveratrol and its glucuronide metabolites, though not sulfate metabolite, reduced triacylglycerol content, although resveratrol was more effective than them. Resveratrol and the three metabolites reduced C/EBPβ mRNA levels. Trans-resveratrol-3-O-sulfate also reduced C/EBP-α, peroxisome proliferator-activated receptor γ (PPAR-γ), and lipoprotein lipase (LPL) expression. In mature adipocytes, resveratrol increased ATGL, CPT-1, deacetylase sirtuin 1 (SIRT-1), and PGC1-α expression. Trans-resveratrol-3-O-glucuronide reduced mRNA levels of FASN and increased those of SIRT-1. Trans-resveratrol-4'-O-glucuronide increased HSL and SIRT-1 mRNA levels. Trans-resveratrol-3-O-sulfate did not change gene expression. The present study shows for the first time the delipidating effect of (i) resveratrol metabolites in maturing pre-adipocytes and (ii) glucuronide metabolites in mature adipocytes. This suggests that both resveratrol and resveratrol metabolites may be involved in the anti-obesity effect of this polyphenol.

Concerted actions of the catechol O-methyltransferase and the cytosolic sulfotransferase SULT1A3 in the metabolism of catecholic drugs

Catecholic drugs had been reported to be metabolized through conjugation reactions, particularly methylation and sulfation. Whether and how these two Phase II conjugation reactions may occur in a concerted manner, however, remained unclear. The current study was designed to investigate the methylation and/or sulfation of five catecholic drugs. Analysis of the spent media of HepG2 cells metabolically labeled with [35S]sulfate in the presence of individual catecholic drugs revealed the presence of two [35S]sulfated metabolites for dopamine, epinephrine, isoproterenol, and isoetharine, but only one [35S]sulfated metabolite for apomorphine. Further analyses using tropolone, a catechol O-methyltransferase (COMT) inhibitor, indicated that one of the two [35S]sulfated metabolites of dopamine, epinephrine, isoproterenol, and isoetharine was a doubly conjugated (methylated and sulfated) product, since its level decreased proportionately with increasing concentrations of tropolone added to the labeling media. Moreover, while the inhibition of methylation resulted in a decrease of the total amount of [35S]sulfated metabolites, sulfation appeared to be capable of compensating the suppressed methylation in the metabolism of these four catecholic drugs. A two-stage enzymatic assay showed the sequential methylation and sulfation of dopamine, epinephrine, isoproterenol, and isoetharine mediated by, respectively, the COMT and the cytosolic sulfotransferase SULT1A3. Collectively, the results from the present study implied the concerted actions of the COMT and SULT1A3 in the metabolism of catecholic drugs.

Validation of determination of plasma metabolites derived from thyme bioactive compounds by improved liquid chromatography coupled to tandem mass spectrometry

In the present study, a selective and sensitive method, based on microelution solid-phase extraction (μSPE) plate and ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS) was validated and applied to determine the plasma metabolites of the bioactive compounds of thyme. For validation process, standards of the more representative components of the phenolic and monoterpene fractions of thyme were spiked in plasma samples and then the quality parameters of the method were studied. Extraction recoveries (%R) of the studied compounds were higher than 75%, and the matrix effect (%ME) was lower than 18%. The LODs ranged from 1 to 65μg/L, except for the thymol sulfate metabolite, which was 240μg/L. This method was then applied for the analysis of rat plasma obtained at different times, from 0 to 6h, after an acute intake of thyme extract (5g/kg body weight). Different thyme metabolites were identified and were mainly derived from rosmarinic acid (coumaric acid sulfate, caffeic acid sulfate, ferulic acid sulfate, hydroxyphenylpropionic acid sulfate, dihydroxyphenylpropionic acid sulfate and hydroxybenzoic acid) and thymol (thymol sulfate and thymol glucuronide). The most abundant thyme metabolites generated were hydroxyphenylpropionic acid sulfate and thymol sulfate, their respective concentrations in plasma being 446 and 8464μM 1h after the intake of the thyme extract.

Recent studies on the bioavailability of dietary (poly)phenolics

HPLC-tandem mass spectrometry is now is now used widely as an the analytical tool in studies on the bioavailability of dietary (poly)phenolics. Recent information on the absorption, disposition, metabolism and excretion of hydroxycinnamates, ellagitannins and various flavonoids that has been obtained in acute human feeding studies with teas, coffee, berries and fruit juices will be summarised. Typically, aglycones released by the action of cytosolic b-glucosidases or lactase phlorizen hydrolase in the small intestine appear in the circulatory system as glucuronide, sulfate and methyl metabolites 1–2h after ingestion. Studies with ileostomists in which ileal fluid collected after ingestion was analysed indicate that substantial amounts of dietary (poly)phenols pass from the small to the large intestine where as a result of the action of the colonic microflora, a diversity of phenolic catabolites are produced. Evidence indicates that these catabolites are absorbed into the circulatory system and can be subjected to some degree of phase II metabolism before excretion in urine in substantial amounts. Of particular interest in this context are valerolactones, derived from flavan-3-ol monomers found in especially high concentrations in green tea and cocoa-based products, and urolithins which are produced in the colon from ellagitannins found in pomegranates and several berries. The potential bioactivity of colonic catabolites will be discussed.

New potential markers for the detection of boldenone misuse

Boldenone is one of the most frequently detected anabolic androgenic steroids in doping control analysis. Boldenone misuse is commonly detected by the identification of the active drug and its main metabolite, 5β-androst-1-en-17β-ol-3-one (BM1), by gas chromatography–mass spectrometry (GC–MS), after previous hydrolysis with β-glucuronidase enzymes, extraction and derivatization steps. However, some cases of endogenous boldenone and BM1 have been reported. Nowadays, when these compounds are detected in urine at low concentrations, isotope ratio mass spectrometry (IRMS) analysis is needed to confirm their exogenous origin. The aim of the present study was to identify boldenone metabolites conjugated with sulphate and to evaluate their potential to improve the detection of boldenone misuse in sports. Boldenone was administered to a healthy volunteer and urine samples were collected up to 56h after administration. After a liquid–liquid extraction with ethyl acetate, urine extracts were analysed by liquid chromatography tandem mass spectrometry (LC–MS/MS) using electrospray ionisation in negative mode by monitoring the transition of m/z 365–350, specific for boldenone sulphate. Boldenone sulphate was identified in the excretion study urine samples and, moreover, another peak with the same transition was observed. Based on the MS/MS behaviour the metabolite was identified as epiboldenone sulphate. The identity was confirmed by isolation of the LC peak, solvolysis and comparison of the retention time and MS/MS spectra with an epiboldenone standard. These sulphated metabolites have not been previously reported in humans and although they account for less than 1% of the administered dose, they were still present in urine when the concentrations of the major metabolites, boldenone and BM1, were at the level of endogenous origin. The sulphated metabolites were also detected in 10 urine samples tested positive to boldenone and BM1 by GC–MS.In order to verify the usefulness of these new metabolites to discriminate between endogenous and exogenous origin of boldenone, four samples containing endogenous boldenone and BM1, confirmed by IRMS, were analysed. In 3 of the 4 samples, neither boldenone sulphate nor epiboldenone sulphate were detected, confirming that these metabolites were mainly detected after exogenous administration of boldenone. In contrast, boldenone sulphate and, in some cases, epiboldenone sulphate were present in samples with low concentrations of exogenous boldenone and BM1. Thus, boldenone and epiboldenone sulphates are additional markers for the exogenous origin of boldenone and they can be used to reduce the number of samples to be analysed by IRMS. In samples with boldenone and BM1 at the concentrations suspicion for endogenous origin, only if boldenone and epiboldenone sulphates are present, further analysis by IRMS will be needed to confirm exogenous origin.

Simultaneous Lc–MS/MS Quantitation of Acetaminophen And Its Glucuronide And Sulfate Metabolites in Human Dried Blood Spot Samples Collected By Subjects in A Pilot Clinical Study

In support of a pilot clinical trial using acetaminophen as the model compound to assess dried blood spot (DBS) sampling as the method for clinical pharmacokinetic sample collection, a novel sensitive LC-MS/MS method was developed and validated for the simultaneous determination of acetaminophen and its major metabolites, acetaminophen glucuronide and sulfate, in human DBS samples collected by subjects via fingerprick. The validated assay dynamic range was from 50.0 to 5000 ng/ml for each compound using a 1/8´´ (3-mm) disc punched from a DBS sample. Baseline separation of the three analytes was achieved to eliminate the possible impact of insource fragmentation of the conjugated metabolites on the analysis of the parent. The overall extraction efficiency was from 61.3 to 78.8% for the three analytes by direct extraction using methanol. The validated method was successfully implemented in the pilot clinical study with the obtained pharmacokinetic parameters in agreement with the values reported in literature.

Dissipation, half-lives, and mass spectrometric identification of endosulfan isomers and the sulfate metabolite on three field-grown vegetables

Endosulfan 3 EC, a mixture of α- and β-stereo isomers, was sprayed on field-grown pepper, melon, and sweet potato plants at the recommended rate of 0.44 kg A.I. acre−1. Plant tissue samples (leaves, fruits, or edible roots) were collected 1 h to 30 days following spraying and analyzed for endosulfan isomers (α- and β-isomers). Analysis of samples was accomplished using a gas chromatograph (GC) equipped with a mass detector in total ion mode. The results indicated the formation of endosulfan sulfate as the major metabolite of endosulfan sulfite and the relatively higher persistence of the β-isomers as compared to the α-isomer. The initial total residues (α- and β-isomers plus endosulfan sulfate) were higher on leaves than on fruits. On pepper and melon fruits, the α-isomer, which is the more toxic to mammals, dissipated faster (T1/2 = 1.22 and 0.95 d, respectively) than the less toxic β-isomer (T1/2 = 3.0 and 2.5 d, respectively). These results confirm the greater loss of the α-isomer compared to the β-isomer, which can ultimately impact endosulfan dissipation in the environment. Additionally, the higher initial residues of endosulfan on pepper and sweet potato leaves should be considered of great importance for timing field operations and the safe entry of harvesters due to the high mammalian toxicity of endosulfan.

Severe diabetes and leptin resistance cause differential hepatic and renal transporter expression in mice

BackgroundType-2 Diabetes is a major health concern in the United States and other Westernized countries, with prevalence increasing yearly. There is a need to better model and predict adverse drug reactions, drug-induced liver injury, and drug efficacy in this population. Because transporters significantly contribute to drug clearance and disposition, it is highly significant to determine whether a severe diabetes phenotype alters drug transporter expression, and whether diabetic mouse models have altered disposition of acetaminophen (APAP) metabolites.ResultsTransporter mRNA and protein expression were quantified in livers and kidneys of adult C57BKS and db/db mice, which have a severe diabetes phenotype due to a lack of a functional leptin receptor. The urinary excretion of acetaminophen-glucuronide, a substrate for multidrug resistance-associated proteins transporters was also determined. The mRNA expression of major uptake transporters, such as organic anion transporting polypeptide Slco1a1 in liver and kidney, 1a4 in liver, and Slc22a7 in kidney was decreased in db/db mice. In contrast, Abcc3 and 4 mRNA and protein expression was more than 2 fold higher in db/db male mouse livers as compared to C57BKS controls. Urine levels of APAP-glucuronide, -sulfate, and N-acetyl cysteine metabolites were higher in db/db mice.ConclusionA severe diabetes phenotype/presentation significantly altered drug transporter expression in liver and kidney, which corresponded with urinary APAP metabolite levels.

Positioning of Antioxidant Quercetin and Its Metabolites in Lipid Bilayer Membranes: Implication for Their Lipid-Peroxidation Inhibition

Among numerous biological activities, natural polyphenols are antioxidants widely distributed in plants capable of inhibiting lipid peroxidation, which belongs to the most serious degenerative cell processes. Positioning of antioxidants in lipid bilayers can provide an insight to the lipid-peroxidation inhibition at the molecular level. This work aims at determining the location and orientation of quercetin and its most representative (glucuronidated, methylated, and sulfated) metabolites in lipid bilayer via molecular dynamic simulations. We show that quercetin derivatives penetrate the lipid bilayer and that the depths of penetration depend on molecular charge and substitutional variations. In the presence of charged substituents (sulfates and glucuronidates), the molecule is pulled toward the lipid bilayer surface. The orientation also depends on substitution as H-bonds are formed between the polar head groups of the bilayer and the (i) OH groups, (ii) sugar, and (iii) sulfate moieties of the antioxidants. As flavonoids and their derivatives are preferentially localized in the lipid bilayer membrane or on the bilayer/water interface, they readily concentrate in a relatively narrow membrane region. Despite the low concentrations of flavonoids in food, their spatial confinement in the membrane greatly enhances their local concentration in this vital region, thus increasing their importance for in vivo biological activities including oxidative stress defense.

Ethanol Sulfation by the Human Cytosolic Sulfotransferases: A Systematic Analysis

Ethyl sulfate, a minor and direct ethanol metabolite in adult human body, has been implicated as a biomarker for alcohol consumption and in utero exposure to ethanol. To understand better the physiological relevance of the sulfation of ethanol, it is important to clarify the cytosolic sulfotransferase (SULT) enzymes that are responsible for ethanol sulfation. The present study aimed to identify the major ethanol-sulfating human SULTs and to investigate the sulfation of ethanol under the metabolic setting. A systematic analysis revealed four ethanol-sulfating SULTs, SULT1A1, SULT1A2, SULT1A3, and SULT1C4, among the eleven human SULT enzymes previously prepared and purified. A metabolic labeling study demonstrated the generation and release of ethyl [(35)S]sulfate in a concentration-dependent manner by HepG2 human hepatoma cells labeled with [(35)S]sulfate in the presence of different concentrations of ethanol. Cytosol or S9 fractions of human lung, liver, and small intestine were examined to verify the presence of ethanol-sulfating activity in vivo. Of the three human organs, the small intestine displayed the highest activity.

Urinary Excretion Kinetics of 3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) and Its Phase I and Phase II Metabolites in Humans following Controlled MDMA Administration

3,4-Methylendioxymethamphetamine (MDMA) is excreted inhuman urine as unchanged drug and phase I and II metabolites. Previous urinary excretion studies after controlled oral MDMA administration have been performed only after conjugate cleavage. Therefore, we investigated intact MDMA glucuronide and sulfate metabolite excretion. We used LC-high-resolution MS and GC-MS to reanalyze blind urine samples from 10 participants receiving 1.0 or 1.6 mg/kg MDMA orally. We determined median C(max),t(max), first and last detection times, and total urinary recovery; calculated ratios of sulfates and glucuronides; and performed in vitro-in vivo correlations. Phase II metabolites of 3,4-dihydroxymethamphetamine (DHMA),4-hydroxy-3-methoxymethamphetamine (HMMA),3,4-dihydroxyamphetamine (DHA), and 4-hydroxy-3-methoxyamphetamine were identified, although only DHMA sulfates, HMMA sulfate, and HMMA glucuronide had substantial abundance. Good correlation was observed for HMMA measured after acid hydrolysis and the sum of unconjugated HMMA, HMMA glucuronide, and HMMA sulfate (R(2) = 0.87). More than 90% of total DHMA and HMMA were excreted as conjugates. The analyte with the longest detection time was HMMA sulfate. Median HMMA sulfate/glucuronide and DHMA 3-sulfate/4-sulfate ratios for the first 24 h were 2.0 and 5.3, respectively, in accordance with previous in vitro calculations from human liver microsomes and cytosol experiments. Human MDMA urinary metabolites are primarily sulfates and glucuronides,with sulfates present in higher concentrations than glucuronides. This new knowledge may lead to improvements in urine MDMA and metabolite analysis in clinical and forensic toxicology, particularly for the performance of direct urine analysis.

Parallel Biotransformation of Tetrabromobisphenol A in Xenopus laevis and Mammals: Xenopus as a Model for Endocrine Perturbation Studies

The flame retardant tetrabromobisphenol A (TBBPA) is a high production flame retardant that interferes with thyroid hormone (TH) signaling. Despite its rapid metabolism in mammals, TBBPA is found in significant amounts in different tissues. Such findings highlight first a need to better understand the effects of TBBPA and its metabolites and second the need to develop models to address these questions experimentally. We used Xenopus laevis tadpoles to follow radiolabeled (14)C-TBBPA uptake and metabolism. Extensive and rapid uptake of radioactivity was observed, tadpoles metabolizing > 94% of (14)C-TBBPA within 8 h. Four metabolites were identified in water and tadpole extracts: TBBPA-glucuronide, TBBPA-glucuronide-sulfate, TBBPA-sulfate, and TBBPA-disulfate. These metabolites are identical to the TBBPA conjugates characterized in mammals, including humans. Most radioactivity (> 75%) was associated with sulfated conjugates. The antithyroid effects of TBBPA and the metabolites were compared using two in vivo measures: tadpole morphology and an in vivo tadpole TH reporter gene assay. Only TBBPA, and not the sulfated metabolites, disrupted thyroid signaling. Moreover, TBBPA treatment did not affect expression of phase II enzymes involved in TH metabolism, suggesting that the antithyroid effects of TBBPA are not due to indirect effects on TH metabolism. Finally, we show that only the parent TBBPA inhibits T3-induced transactivation in cells expressing human, zebrafish, or X. laevis TH receptor, TRα. We conclude, first, that perturbation of thyroid signaling by TBBPA is likely due to rapid direct action of the parent compound, and second, that Xenopus is an excellent vertebrate model for biotransformation studies, displaying homologous pathways to mammals.

Determination of resveratrol and its sulfate and glucuronide metabolites in plasma by LC–MS/MS and their pharmacokinetics in dogs

An analytical approach for the determination of trans-resveratrol (3,5,4′-trihydroxy- trans-stilbene) and its glucuronide and sulfate conjugates in dog plasma by LC–MS/MS (without enzymatic hydrolysis of the conjugates) was validated to support pre-clinical toxicological and pharmacological studies. The approach required two independent sample extractions and consequent instrument runs. Samples for resveratrol determination were prepared by protein precipitation with acetonitrile; acetonitrile–methanol was used instead for resveratrol metabolites. Chromatographic separation was performed using a C18 column (30 mm × 2.0 mm) at a flow rate of 0.25 mL/min. For resveratrol the mobile phase consisted of A: 5 mM ammonium acetate in water–isopropanol (98:2, v/v) and B: methanol–isopropanol (98:2, v/v) and for metabolites the mobile phase was modified as follows: A: 0.1% (v/v) formic acid in water and B: 0.1% (v/v) formic acid in acetonitrile. Total run time was 12 min for each run with retention times of about 4–5 min for all analytes. A turbo ion spray source was used operating in negative mode for resveratrol and resveratrol sulfate and in positive mode for resveratrol glucuronide. Calibration curves were linear from 5 to 1000 ng/mL for resveratrol and its glucuronide, and 10–2000 ng/mL for resveratrol sulfate. Linearity was assessed using the internal standard method for resveratrol and the external standard method for the metabolites. Method accuracy was 90–112% of the true value for all analytes with precision of 9% RSD or less for all validation experiments. The validated method was applied to a preclinical toxicology study in dogs after oral administration (200–1200 mg/kg) of the agent. Peak plasma resveratrol concentration ( C max) for most animals was observed within 1–5 h of dosing, with group mean values in the 1.7–9.9 μg/mL (7.5–43 μM) range. Area under the plasma concentration–time curve (AUC) mean values for resveratrol ranged from 3.6 to 44 h μg/mL for all study groups and were generally proportional to the dose, with no consistent statistically significant changes observed for gender or number of doses. Mean molecular-weight adjusted ratios of resveratrol metabolites to resveratrol for AUC ranged from 1 to 9 for resveratrol glucuronide and from 2 to 11 for resveratrol sulfate.